Mycologia, 102(6), 2010, pp. 1350–1368. DOI: 10.3852/10-006 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897
A six locus phylogeny reveals high species diversity in Botryosphaeriaceae from California almond Patrik Inderbitzin
established species and one was morphologically distinct from its closest relatives, Neof. andinum and Neof. arbuti, as well as from the more than 190 described species of Fusicoccum and Neofusicoccum, and thus was described as the new species, Neof. nonquaesitum. Evidence for cryptic speciation was found in B. dothidea, Neof. ribis and Spencermartinsia viticola. Botryosphaeria dothidea and Neof. ribis comprised lineages that formed the morphologically distinct Dichomera anamorph not found in any other isolates recognized as B. dothidea and Neof. ribis. An S. viticola isolate from California was phylogenetically divergent and had conidia that differed morphologically from the type. Neofusicoccum parvum was diverse but lacked any morphological features correlating with molecular diversity. Phylogenetic analyses of combinations of datasets showed that pooled analyses of all six datasets resulted in the highest number of supported branches, suggesting that addition of more data might yet improve phylogenetic resolution. Key words: D. mutila, Dich. eucalypti, Dich. versiformis, Do. iberica, Lasiodiplodia theobromae, Neoscytalidium dimidiatum, Neof. australe, Neof. luteum
Department of Plant Pathology, University of California, One Shields Avenue, Davis, California 95616, and Kearney Agricultural Center, 9240 South Riverbend Avenue, Parlier, California 93648
Richard M. Bostock Florent P. Trouillas Department of Plant Pathology, University of California, One Shields Avenue, Davis, California 95616
Themis J. Michailides1 Department of Plant Pathology, University of California, One Shields Avenue, Davis, California 95616, and Kearney Agricultural Center, 9240 South Riverbend Avenue, Parlier, California 93648
Abstract: Botryosphaeriaceae are important pathogens on a variety of woody hosts, including almond, a major crop in California. Almond is susceptible to Botryosphaeria dothidea that forms band cankers on almond trunks, and the same fungus was also isolated from cankers of the canopy. To study the diversity and host range of B. dothidea and allied species from almond we used 132 isolates from 36 plant hosts from five continents, including 45 strains from almond in California. Species were identified by comparison to 13 ex-type strains with phylogenetic analyses based on six loci, including the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene repeat and portions of the coding genes elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein, histone-3 and betatubulin. Seven species were found from almond: Botryosphaeria dothidea, Neofusicoccum parvum, Neof. mediterraneum, Neof. nonquaesitum, Diplodia seriata and Macrophomina phaseolina were identified from band cankers, and B. dothidea, Neof. mediterraneum, Neof. parvum and Dothiorella sarmentorum from canopy cankers. All were capable of inducing cankers on inoculated almond branches in the field. All species found on almond also occurred on other hosts, suggesting that infected vegetation adjacent to almond orchards could serve as source of inoculum of virulent almond strains. Of the 19 monophyletic groups obtained at the species level, 13 contained ex-type strains, five were morphologically similar to
INTRODUCTION
The ascomycete family Botryosphaeriaceae plays an important role in the ecology of woody plants. Family members are frequently isolated from non-symptomatic as well as diseased tissues (Slippers and Wingfield 2007), and in California the single morphological species Botryosphaeria dothidea has been found on at least 42 hosts (Michailides and Morgan 2004). Diseases caused by species of Botryosphaeriaceae can be detrimental to crops and in California have been reported from almond (English et al. 1966), ´ rbez-Torres and avocado (Horne 1932), grapevine (U ´ Gubler 2007, Urbez-Torres et al. 2006), fig (T.J. Michailides unpubl), pistachio (Michailides 1991, Rice et al. 1985), walnut (Michailides and Morgan 2004), as well as from native chaparral where Botryosphaeria branch dieback has been linked to forest fires (Brooks and Ferrin 1994). Many more hosts are known worldwide. Almond is one of California’s most important crops, and it is susceptible to band canker caused by B. dothidea. Band canker first was found in California in 1959 and is characterized by narrow bands of diseased tissue (cankers) that extend around the
Submitted 5 Jan 2010; accepted for publication 5 Apr 2010. 1 Corresponding author. E-mail:
[email protected]
1350
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND trunk (English et al. 1966). Usually 3–4 y old trees are affected and suffer losses, but in general cankers disappear naturally and the trees survive. The canker agent has been identified as B. dothidea based on morphological and serological analyses (English et al. 1975). The same fungus also has been isolated from cankers of the canopy since at least the early 1980s (R.M. Bostock unpubl). Cankers of the canopy affect small branches and fruit, thereby reducing yields. Considerable changes have occurred in Botryosphaeria taxonomy in recent years. Genus Botryosphaeria has been restricted to a small monophyletic group containing B. dothidea, the type of Botryosphaeria. Taxa falling into other groups have been transferred to new genera, such as Neofusicoccum and Spencermartinsia, or are now cited by their anamorph names only, such as Diplodia, Dothiorella and Lasiodiplodia (Crous et al. 2006, Phillips et al. 2008). The taxonomy of Botryosphaeriaceae anamorphs is complex; more than a dozen anamorph genera are associated with Botryosphaeriaceae (Denman et al. 2000). Anamorphs can be coelo- or hyphomycetes and are separated by conidial pigmentation, ontogeny, septation and morphology. Commonly found and of relevance here are pycnidia genera, including Fusicoccum and Neofusicoccum, that have fusiform, hyaline conidia that may become pigmented and multiseptate in age or after discharge from the pycnidia (Crous and Palm 1999, Crous et al. 2006). Diplodia conidia are oblong to ellipsoid, hyaline at first and then turn brown, as opposed to Dothiorella conidia that become pigmented while attached to the conidiophore (Phillips et al. 2005a). Lasiodiplodia conidia are brown and striate (Alves et al. 2008), and Dichomera conidia are brown with both transverse and longitudinal septa. Macrophomina conidia are hyaline, with long, unfurling appendages (Crous et al. 2006, Sutton 1980). Neoscytalidium dimidiatum has both a coelomycete state with pigmented conidia, accompanied by a phragmosporous, pigmented hyphomycete synanamorph (Crous et al. 2006, Sutton and Dyko 1989). The asexual state of Botryosphaeria dothidea, the almond band canker pathogen, is Fusicoccum aesculi Corda that is found more frequently in the field than its sexual state, and is similar in morphology to Neofusicoccum. Evidence based on molecular data from another disease in California attributed to B. dothidea, pistachio panicle and shoot blight, showed that the causal agent is distinct from B. dothidea, even though the two were indistinguishable morphologically (Smith et al. 2001a). In almond similar studies have not been done. New species are continually being found in Botryosphaeriaceae (Luque et al. 2005; Phillips et al. 2005a;
1351
Phillips et al. 2002; Slippers et al. 2004a, b; Smith et al. 2001b), and established species are being emended. Pavlic et al. (2009a, b) used five genes in an investigation of the Neof. parvum and Neof. ribis species complex and found three new species. From eucalypts in Australia Barber et al. (2005) found Fusicoccum strains producing Dichomera synanamorphs that had identical or similar ITS sequences to B. dothidea, Neof. ribis, Neof. parvum and Neof. australe. None of these species were known previously to form any Dichomera synanamorphs. The goals of this study were to investigate the diversity of Botryosphaeriaceae species on almond using a six gene multigene phylogenetic approach and to include ex-type strains whenever possible. We also were interested in the host ranges and geographic distributions of the species, as well as in species limits and intraspecific variation. MATERIALS AND METHODS
Taxon sampling and origins of fungal strains.— Our sampling was centered on isolates from California almond. To provide context isolates from other hosts and geographic locations also were included. We used a total of 132 strains (TABLE I), 109 collected in this study and 23 from culture collections. The cultures originated from at least 36 plant host species from 12 countries and five continents, including 105 from USA and 95 from California. Of the California isolates 45 were from almond, 10 from pistachio, eight from walnut and 32 from an additional 16 hosts. Thirteen ex-type strains were included. Fungal isolation and culturing.—Symptomatic plant tissue, including pieces of bark from band-cankered trunks, blighted shoots and peduncles, as well as wood chips from cankers, was returned to the laboratory and examined for the presence of pycnidia and ascomata. Fungal cultures were derived from single conidia or ascospores or, when fungal fruiting structures were absent, by plating surfacesterilized plant tissue (10% bleach 3 min) on acidified PDA (lactic acid 0.065% v/v) and subsequent single conidium purification according to Ma et al. (2001b), with the following alterations. Pistachio, cork oak or peach leaves were autoclaved twice in sterile water and placed on a thin layer of half strength PDA agar covering approximately half a Petri plate. After inoculation plates were left unsealed and incubated in plastic containers on a laboratory bench under continuous artificial light and natural light during daytime. To promote conidiation container lids were left ajar once mycelial growth covered the agar surface. Microscopy, measurements and color descriptions.—Photographs were taken on a Nikon Microphot-SA or a Leica DM5000B microscope, and measurements were made with SpotAdvanced software (Diagnostic Instruments Inc., Sterling Heights, Michigan). Mean and standard deviations were determined for conidia lengths, widths and length to width ratios. Colors were according to Rayner (1970).
Species
Dichomera versiformis Z.Q. Yuan, Wardlaw & C. Mohammede,f Diplodia mutila (Fr.) Mont. Diplodia mutila Diplodia mutila Diplodia mutila Diplodia seriata de Not. Diplodia seriata Diplodia seriata Diplodia seriata Diplodia seriata Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alvesg,h
Dichomera eucalypti f
Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Dichomera eucalypti (G. Winter) B. Suttonf Dichomera eucalypti e,f
Botryosphaeria dothideaf
PD46, 2900 PD61, 3208 PD73, 9 PD75, 8 PD13, 266 PD34, 3381 PD47, 3594 PD50, 3348 PD272, 53 PD257, CBS 115041, LISE 94944
Pistacia vera Prunus dulcis Olea europea Prunus dulcis Rubus sp. Prunus dulcis Prunus dulcis Prunus dulcis Prunus sp.
PD27, 222 PD33, 3657 PD58, 686 PD68, 3623 PD71, 131 PD107, 809 PD122, A2.1 PD146, A27 PD255, CMW 8000, PREM 57372 PD296, WAC 12404, WA7 PD313, B. dothidea 1 PD314, B. dothidea 2 PD315, B. dothidea 3 PD290, WAC 12398, Bot6 PD293, WAC 12401, VIC1, VPRI 31987, IMI 59162 PD294, WAC 12402, VIC2 PD295, WAC 12403, VIC3, VPRI 31988 ? Persea americana Ilex sp. Ilex sp. Prunus persica Prunus dulcis Juglans regia Prunus dulcis Pistacia vera Quercus ilex
E. camaldulensis
shoot fruit branch branch shoot trunk bark shoot band canker shoot twig
leaf
—
conidium conidium conidium conidium conidium conidium conidium conidium
—
—
—
leaf
E. pauciflora
leaf
mycelium mycelium mycelium —
fruit fruit fruit twig
Malus domestica Malus domestica Malus domestica Eucalyptus diversicolor
E. camaldulensis
—
conidium conidium ascospore ascospore conidium conidium mycelium conidium —
9/1990 6/2004 12/1988 12/1988 3/2006 7/2004 4/2005 8/2004 2/1999 12/2009
—
—
—
2007 2007 2007 2001
3/2003
7/2005 8/2005 5/2000 8/2005 4/2000 8/2005 5/2007 5/2007 2000
8/2005
canopy canker conidium rachis fruit peduncle shoot band canker cane canopy canker canopy canker band canker —
Collection date
Inoculum sourcea
Substrate
stem
E. calophylla
Prunus dulcis
Host
PD4, 3626
Identifiers
One hundred thirty-two isolates were used in this study
Botryosphaeria dothidea (Moug.) Ces. & de Not. Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidea Botryosphaeria dothidead,e
TABLE I.
? TJM, PI TJM/TJM, TJM/TJM, TJM/TJM, TJM/TJM, TJM/TJM, TJM/TJM, TJM/TJM, J. Luque
PI PI PI PI PI PI PI
P. A. Barber
G. Whyte
T. Sutton T. Sutton T. Sutton T. Burgess, K.2L. Goei P. J. Keane
T. Paap
OR, USA Ventura Co., CA, USA CA, USA CA, USA Fresno Co., CA, USA Kern Co., CA, USA Merced Co., CA, USA Colusa Co., CA, USA Kern Co., CA, USA Spain
Australia
Australia
Australia
Clayton, NC, USA Clayton, NC, USA Clayton, NC, USA Australia
Australia
Imathias, Greece Colusa Co., CA, USA Glenn Co., CA, USA Butte Co., CA, USA Glenn Co., CA, USA Glenn Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Switzerland
Glenn Co., CA, USA
TJMb/TJM, PIc TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM, HR, PI/PI TJM, HR, PI/PI B. Slippers
Location
Collector/isolator
1352 MYCOLOGIA
Species
Continued Identifiers
Pistacia vera Pistacia vera Prunus dulcis Eucalyptus sp.
Arbutus menziesii
Arbutus menziesii Arbutus menziesii
PD161 PD270, AW2 PD112, A28.1
PD252, CMW 13455, CBS 117453
PD244, Arbutus 1
PD245, Arbutus 3 PD281, AR 4014, BPI 863597 PD282, AR 4036, CBS 116131 PD283, AR 4100, CBS 117089, UW22, BPI 863937 PD284, 1.2.1
—
conidium mycelium mycelium
— conidium
—
—
—
—
—
twig
Vaccinum corymbosum cv. Duke
canopy canker mycelium
—
—
canopy canker mycelium — —
canopy canker mycelium
—
branch buds band canker
— shoot
Acacia sp.
Arbutus menziesii
Arbutus menziesii
Arbutus menziesii
Ulmus sp. Prunus domesticus
12/2005
—
5/2008
10/1998
10/2003
3/2008 10/1996
3/2008
—
9/2007 5/2008 5/2007
8/1956 10/1997
8/2006
canopy canker conidium
Prunus dulcis
Collection date 8/2006
Inoculum sourcea
canopy canker conidium
Substrate
Prunus dulcis
Host
PD280, 63581b PD20, 69
Neofusicoccum australe PD253, CMW6838, PREM 57589 Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillipsl,h Neofusicoccum australe PD298, B1205
Neofusicoccum arbuti
Neofusicoccum arbuti j
Neofusicoccum arbuti h,k
Dothiorella sarmentorumf,g Lasiodiplodia theobromae (Pat.) Griffon & Maubl. Lasiodiplodia theobromae Lasiodiplodia theobromae Macrophomina phaseolina (Tassi) Goid. Neofusicoccum andinum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf.i,h Neofusicoccum arbuti (D.F. Farr & M. Elliott) Crous, Slippers & A.J.L. Phillips Neofusicoccum arbuti Neofusicoccum arbuti j
Dothiorella sarmentorum (Fr.) PD78, 3797 A.J.L. Phillips, A. Alves & J. Luque Dothiorella sarmentorum PD79, 3795
TABLE I.
Stanislaus Co., CA, USA Great Britain Butte Co., CA, USA
Stanislaus Co., CA, USA
Location
CA, USA
WA, USA
King Co., WA, USA WA, USA
King Co., WA, USA
Venezuela
E.X. Bricen ˜ o, J.G. Espinoza, B.A. Latorre/ E.X. Bricen ˜o
Rapel, VI Region, Chile
C. M. Monterey Co., CA, USA McCormick/PI M. J. Wingfield Australia
M. Elliott/A. Rossman M. Elliott
M. Elliott/PI M. Elliott
M. Elliott/PI
S. Mohali
J. Cook/TJM, PI Wilcox Co., AZ, USA TJM/TJM, PI Madera Co., CA, USA TJM, HR, PI/PI Butte Co., CA, USA
E. A. Ellis TJM/TJM, PI
TJM/TJM, PI
TJM/TJM, PI
Collector/isolator
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND 1353
PD55, 2953 PD56, 3271 PD67, 7 PD82, 692 PD84, 18.1 PD87, A41.1 PD88, A41.2 PD108, 3449 PD115, A2.2 PD120, A10.4 PD123, A8.3 PD125, A4.1 PD127, A24.1 PD129, A43.2 PD132, A26.1 PD138, A24.2 PD139, A4.2 PD141, A45.8 PD143, A4.4 PD144, A45.3 PD145, A26.2
mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum mediterraneum
Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum
PD285, CBS 110299, LISE 94070 PD1, 12
Identifiers
PD2, 86 PD5, 3722 PD9, 3354 PD14, 99 PD16, 691 PD24, 83 PD25, 95 PD30, 3718 PD48, 3483 PD49, 3227 PD53
Species
Continued
Neofusicoccum luteum Pennycook & Samuelsh,m Neofusicoccum mediterraneum Crous, M.J. Wingf. & A.J.L. Phillips Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum
TABLE I.
conidium
—
Inoculum sourcea
band canker shoot fruit cane branch branch branch canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker canopy canker
conidium conidium conidium conidium conidium conidium conidium ? mycelium mycelium mycelium mycelium mycelium mycelium mycelium mycelium mycelium mycelium mycelium mycelium mycelium
shoot conidium shoot conidium shoot conidium shoot conidium cane conidium shoot conidium shoot conidium rachis conidium canopy canker conidium canopy canker conidium shoot conidium
shoot
Juniperus sp.
Eucalyptus sp. Juglans regia Fortunella sp. Eucalyptus sp. Rubus sp. Juglans regia Citrus sp. Pistacia vera Prunus dulcis Prunus dulcis Sequoiadendron giganteum Prunus dulcis Fortunella sp. Persea americana Rubus sp. Juglans regia Juglans regia Juglans regia Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis
cane
Substrate
Vitis vinifera
Host
1/2004 5/2004 ? 4/2000 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007 5/2007
7/1999 11/2005 7/2004 11/1999 4/2000 7/1999 11/1999 10/2005 9/2004 6/2004 4/2007
6/1999
3/1996
Collection date
TJM/TJM, PI TJM/TJM, PI TJM, PI TJM/TJM, PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM/TJM, PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI
TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM, PI/PI
TJM/TJM, PI
A. J. L. Phillips
Collector/isolator
Fresno Co., CA, USA Colusa Co., CA, USA Riverside, CA, USA Glenn Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Colusa Co., CA, USA Colusa Co., CA, USA Colusa Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA
Fresno Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Fresno Co., CA, USA Glenn Co., CA, USA Fresno Co., CA, USA Fresno Co., CA, USA Butte Co., CA, USA Merced Co., CA, USA Colusa Co., CA, USA Fresno Co., CA, USA
Fresno Co., CA, USA
Portugal
Location
1354 MYCOLOGIA
Prunus dulcis
Prunus dulcis Prunus dulcis Umbellularia californica Umbellularia californica Umbellularia californica Umbellularia californica Vaccinum corymbosum cv. Elliot
Vaccinum corymbosum cv. Brigitta
Umbellularia californica Pistacia vera
PD86, A9
PD90, A42 PD149, A44.1 PD246, L3IE2 PD247, L3IE5 PD248, L5E2 PD249, L7IE3 PD301, B62207
PD302, B02207
PD484, L3IE1 PD6, 220
Neofusicoccum nonquaesitum
Neofusicoccum nonquaesitum Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips
Prunus dulcis Fraxinus sp. Pistacia vera Pistacia vera Pistacia vera Pistacia vera Pistacia vera Pistacia vera Pistacia vera Olea europea Eucalyptus sp.
Neofusicoccum nonquaesitum Inderbitzin, F.P. Trouillas, R.M. Bostock et T.J. Michailides Neofusicoccum nonquaesitum Neofusicoccum nonquaesitum Neofusicoccum nonquaesitum Neofusicoccum nonquaesitum Neofusicoccum nonquaesitum Neofusicoccum nonquaesitum Neofusicoccum nonquaesitum
A7.1 D1.1 45 56 57 65 315 318 833 CBS 121558 CBS 121718
Host
PD147, PD155, PD271, PD273, PD274, PD275, PD276, PD278, PD279, PD311, PD312,
Identifiers
Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneum Neofusicoccum mediterraneumn Neofusicoccum mediterraneumh,n
Species
TABLE I. Continued
twig leaf
twig
band canker band canker twig twig twig twig twig
band canker
band canker branch fruit shoot fruit shoot fruit fruit shoot drupe branch/leaf
Substrate
mycelium conidium
—
conidium conidium mycelium mycelium mycelium mycelium —
conidium
conidium conidium mycelium mycelium mycelium mycelium mycelium mycelium mycelium — —
Inoculum sourcea
11/2004 7/2005
11/2006
5/2007 5/2007 11/2004 11/2004 11/2004 11/2004 11/2006
5/2007
5/2007 6/2007 1/1999 2/1999 2/1999 ? 4/1990 7/1990 9/2007 11/2004 6/2006
Collection date
TJM, HR, PI/PI TJM, HR, PI/PI F. P. Trouillas F. P. Trouillas F. P. Trouillas F. P. Trouillas E.X. Bricen ˜ o, J.G. Espinoza, B.A. Latorre/ J.G. Espinoza E.X. Bricen ˜ o, J.G. Espinoza, B.A. Latorre/ J.G. Espinoza F. P. Trouillas TJM/TJM, PI
TJM, HR, PI/PI TJM/PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI C. Lazzizera P.W. Crous, M.J. Wingfield, A.J.L. Phillips TJM, HR, PI/PI
Collector/isolator
Napa Co., CA, USA Imathias, Greece
Nancagua, VI Region, Chile
Butte Co., CA, USA Butte Co., CA, USA Napa Co., CA, USA Napa Co., CA, USA Napa Co., CA, USA Napa Co., CA, USA Rı´o Negro, Osorno, X Region, Chile
Colusa Co., CA, USA
Colusa Co., CA, USA Fresno Co., CA, USA Kern Co., CA, USA Madera Co., CA, USA Madera Co., CA, USA Madera Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Kern Co., CA, USA Italy Greece
Location
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND 1355
Neofusicoccum ribis
Neofusicoccum ribisf
Neofusicoccum parvumh,p Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillipsd,h Neofusicoccum ribisf
Neofusicoccum parvumo
parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvum parvumo
Species
Continued
Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum Neofusicoccum
TABLE I.
PD288, WAC 12395, FNQ58B PD289, WAC 12396, FNQ27C PD299, B1206
PD17, 3621 PD18, 3721 PD29, 3723 PD39, 3656 PD43, 3678 PD57, 3622 PD59, 3475 PD62, 217 PD65, 3677 PD81, 3694 PD92, A25 PD93, A5.2 PD94, A4 PD106, 661 PD140, A6.1 PD142, A30.9 PD148, A29.2 PD250, Bot 19, CMW 10123 PD251, Bot 21, CMW 10122 PD286, ATCC 58191 PD254, CMW 7772, PREM 57368
Identifiers
E. grandis 3 E. camaldulensis Vaccinum corymbosum 3 darrowi cv. Mysti
Eucalyptus pellita
Populus nigra Ribes sp.
Eucalyptus grandis
Prunus dulcis Juglans regia Juglans regia Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Pistacia vera Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Prunus dulcis Eucalyptus smithii
Host
— —
twig
—
— —
—
conidium ascospore conidium conidium ascospore conidium conidium conidium ascospore conidium conidium conidium conidium conidium mycelium mycelium conidium —
Inoculum sourcea
stem
stem
branch —
—
band canker shoot shoot fruit peduncle band canker band canker canopy canker shoot band canker band canker band canker band canker band canker canopy canker canopy canker canopy canker band canker —
Substrate
5/2006
2003
2003
12/1981 2000
—
8/2005 11/2005 11/2005 8/2005 10/2005 8/2005 2/2005 7/2005 10/2005 10/2005 5/2007 5/2007 5/2007 7/2006 5/2007 5/2007 5/2007 —
Collection date
South Africa
Butte Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Greece Butte Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Colusa Co., CA, USA Butte Co., CA, USA Butte Co., CA, USA South Africa
Location
T. Burgess, G. Pegg T. Burgess, G. Pegg E.X. Bricen ˜ o, J.G. Espinoza, B.A. Latorre/ E.X. Bricen ˜o
Santiago, Metropolitan region, Chile
Australia
Australia
S. R. Pennycook New Zealand G. Hudler NY, USA
H. Smith
TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM/TJM, PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI TJM/TJM, PI TJM, HR, PI/PI TJM, HR, PI/PI TJM, HR, PI/PI H. Smith
Collector/isolator
1356 MYCOLOGIA
Ficus carica Ficus carica Citrus sp.
Vitis vinifera Malus domestica Malus domestica
PD104, 3C2, 3CO2 PD105, 3C7, 3CO7 PD74, 2959
PD258, CBS 117009, LISE 95177 PD23, 79
PD80, 78
fruit
fruit
cane
conidium
conidium
—
conidium conidium conidium
conidium
limb limb limb shoot
—
Inoculum sourcea
twig
Substrate
4/1999
4/1999
12/2004
5/2006 5/2006 1/2004
11/2005
5/2006
Collection date
TJM/TJM, PI
J. Luque, S. Martos TJM/TJM, PI
TJM, PI TJM, PI TJM/TJM, PI
E.X. Bricen ˜ o, J.G. Espinoza, B.A. Latorre/ E.X. Bricen ˜o TJM, PI
Collector/isolator
Fresno Co., CA, USA
Fresno Co., CA, USA
Spain
Madera Co., CA, USA Madera Co., CA, USA Fresno Co., CA, USA
Madera Co., CA, USA
Rapel, VI Region, Chile
Location
b
Inoculation source (viz. ascospore, conidium or mycelium) refers to the fungal tissue used for initial culturing, all strains were subsequently single conidia purified. T.J. Michailides. c P. Inderbitzin. d Slippers et al. (2004a). e Ex-epitype. f Barber et al. (2005). g Phillips et al. (2005a). h Ex-type. i Mohali et al. (2006). j Farr DF and Rossman AY. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved 28 Jun 2009 from http://nt.ars-grin.gov/ fungaldatabases/. k Farr et al. (2005). l Slippers et al. (2004b). m Phillips et al. (2002). n Crous et al. (2007). o Smith et al. (2001b). p Pennycook and Samuels (1985). q Luque et al. (2005).
a
Sphaeropsis sapinea (Fr.) Dyko & B. Sutton Sphaeropsis sapinea
Ficus carica
PD103, 2D3, 2DO3
Neoscytalidium dimidiatum (Penz.) Crous & Slippers Neoscytalidium dimidiatum Neoscytalidium dimidiatum Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L. Phillips, A. Alves & Crous Spencermartinsia viticolah,q
Host Vaccinum corymbosum cv. Duke
Identifiers
PD300, B4.2206
Species
Continued
Neofusicoccum ribis
TABLE I.
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND 1357
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MYCOLOGIA
Virulence assays.—Twenty-four isolates were used for field inoculations on mature almond trees. These were selected randomly from each species and were B. dothidea strains PD4, PD33, PD71 and PD122, Dip. seriata strains PD50 and PD80, Neof. parvum strains PD18, PD29, PD92, PD93, PD140 and PD148, Do. sarmentorum strains PD78 and PD79, Neof. nonquaesitum strains PD86, PD90 and PD149, M. phaseolina strain PD112, and Neof. mediterraneum strains PD30, PD53, PD56, PD84, PD88 and PD147. Inoculations were performed primarily on 1 y old branches. The bark was removed with a 5 mm diam cork borer, and an agar disk with mycelium was placed on the circular lesion with a sterile needle and sealed with Parafilm. Five replicates were performed for each isolate as well as a control lacking mycelium; replicates were assigned randomly to branches across 30 mature almond trees at the Kearney Ag Center, Parlier, California. Branches were harvested 15 Oct 2007, 54 d after inoculation, and returned to the laboratory for evaluation. The progression of the canker along the branch was measured, and blighting symptoms of inoculated branches were noted. Statistical analyses were performed with JMP 8.0 (SAS Institute Inc., Cary, North Carolina). DNA extraction, PCR amplification and DNA sequencing.— Mycelium was scraped off a culture growing on a PDA plate and DNA extracted with the FastDNA Kit in conjunction with the FastPrep Instrument (MP Biomedicals, Irvine, California). Buffer CLS-Y was used, and grinding intensity was set to 4.5 for 30 s. PCR was performed with Promega PCR MasterMix (Promega Corp., Madison, Wisconsin) in a 25 mL reaction volume according to the manufacturer’s instructions. The PCR program consisted of a 2 min initial denaturation step at 94 C, 32 cycles of 10 s at 94 C, 20 s at the primer pair dependent annealing temperature and 1 min at 72 C, followed by a final extension of 7 min at 72 C. PCR bands too weak for sequencing were re-amplified according to Berbee (1999), with the following alterations. Bands were cut from the gel, melted in 50 mL water, diluted 10-fold in H2O, and used for PCR amplification under standard conditions with 10 mL of the diluted sample as template. PCR products were purified by sodium acetate precipitation. DNA sequences were determined at the UC DNA Sequencing Facility, UC Davis, with ABI BigDye Terminator 3.1 Cycle Sequencing chemistry on an ABI 3730 Capillary Electrophoresis Genetic Analyzer (Applied Biosystems, Foster City, California). Loci and primers used.—Six loci were targeted for PCR amplification and DNA sequencing in all isolates. These were the ribosomal internal transcribed spacer region (ITS) and parts of five protein-coding genes: elongation factor-1 alpha (EF1-alpha), glyceraldehyde-3-phosphate dehydrogenase (GPD), histone-3 (HIS), heat shock protein (HSP) and beta-tubulin (TUB). Primers used for PCR and DNA sequencing were designed based on sequences from GenBank or sequences generated in this study or were taken from the literature. The ITS region was PCR amplified with primers ITS1-F (Gardes and Bruns 1993) and ITS4 and sequenced with ITS5 and ITS4 (White et al. 1990). EF was amplified with EF446f (Inderbitzin et al. 2005) and EF1035r (59-GGT GAT ACC ACG CTC ACG CTC-
39) at an annealing temperature of 55 C, targeting approx. 440 bp, and sequenced with the same primers. Primers for the remaining loci were newly designed for this study. GPD was amplified with GPD1if (59-ATC GTC TTC CGC AAC GCG-39) and GPD3ir (59-GAT GTT GGT AGC AGC GGT ACG-39) at 57 C, targeting approx. 730 bp, and sequenced with the same primers or occasionally with internal the GPD1i2f (59-GTC TTC CGC AAC GCG TAA GT -39) and GPD3i2r (59- TAC GGC CAC CAC GCC AGT CC -39). HIS was amplified primarily with HisF3 (59-CAA GCA GAC TGC CCG TAA G-39) and HisR (59-GGC GAG CTG GAT GTC CTT-39) at 54 C targeting approx. 550 bp and sequenced with the same primers. The external HisF (59-ATG GCY MGN ACY AAG CAG AC-39) was used initially instead of HisF3. HSP was amplified with HspF3 (59-CAC AAG GTA CGA CTC CAT TG-39) and HspR (59- ACA GTG GCG GTR GTG GTA CCG T -39) at 52 C, targeting approx. 430 bp and sequenced with the same primers. The external HspF (59CAG CAG CAA CGH TTY GCY CAC -3) initially was used instead of HspF. TUB was amplified with Btf (59-GTT CAT CTC CAG ACC GGT CA-39) and Btr2 (59-AGC TCG GCA CCC TCA GTG T-39) at 52 C, targeting approx. 520 bp and sequenced with the same primers. Internal Btf2 (59-CCA GAC CGG TCA ATG CGT AAG T-39) occasionally was substituted for Btf. Phylogenetic analyses.—Two algorithms were used. Single locus datasets were analyzed under the maximum parsimony criterion with PAUP 4.0b 10 (Swofford 2002). The combined six locus dataset was analyzed with parsimony, as well as MrBayes 3.0b4 (Ronquist and Huelsenbeck 2003), implementing a Bayesian approach to inferring phylogenies. Combinations of three, four and five datasets were analyzed with parsimony to determine the influence of dataset combinations and amounts of data on tree topology and support. Most parsimonious trees were inferred with 30 random addition replicates. Otherwise default settings were used, including treating insertion/deletion gaps as missing data. Bootstrap support values were based on 500 replicates. Bayesian analyses were performed with default settings, running four chains over 106 generations sampling each 100th tree. The first 103 of the 104 saved trees were discarded, and the consensus tree was based on the remaining 9000 trees. Bayesian analyses implemented an optimal model of DNA sequence evolution determined using Modeltest 3.7 (Posada and Crandall 1998). All analyses were run with a single representative of each allele or haplotype to speed up the process. RESULTS
Conidial measurements of Botryosphaeria and Neofusicoccum strains from almond.—Dimensions are from conidia obtained in culture on pistachio, cork oak and peach leaf agar. B. dothidea: strains PD4, PD33: 25.6 6 2.8 3 6.5 6 1.0 mm (l/w 5 4.0 6 0.7, n 5 52); Neof. mediterraneum strains PD108, 123, 143, 147: 22.0 6 2.0 3 7.3 6 0.7 mm (l/w 5 3.0 6 0.4, n 5
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND
FIG. 1. Mean canker lengths after 54 d caused by Botryosphaeriaceae species on almond branches inoculated with mycelial plugs. Bars topped by letters indicate significant differences with a Tukey-Kramer test for multiple comparisons of means. These numbers of isolates per species were used with five replicates each: Neof. nonquaesitum (3), Neof. parvum (6), M. phaseolina (1), Neof. mediterraneum (6), B. dothidea (4), Dip. seriata (2), and Do. sarmentorum (2).
164); Neof. parvum strains PD17, PD39, PD57, PD59, PD65, PD94, PD148: 17.8 6 2.7 3 6.5 6 0.6 mm (l/w 5 2.8 6 0.6, n 5 317); Neof. nonquaesitum strains PD86, PD90, PD149: 24.3 6 2.8 3 7.6 6 1.0 mm (l/w 5 3.2 6 0.4, n 5 397). Virulence assays.—All species were capable of forming cankers on inoculated almond branches (FIG. 1), but the mean canker lengths differed significantly between isolates (P , 0.0001; Wilcoxon rank-sum test). Neofusicoccum nonquaesitum and Neof. parvum were significantly more virulent than the remaining species including B. dothidea; M. phaseolina was not significantly more virulent than any other species (TukeyKramer test for multiple comparison of means). Blighting of inoculated branches was observed for all species for at least one isolate, except M. phaseolina and Do. sarmentorum where no blighting was observed. The only species where all isolates caused blighting in all replicates was Neof. nonquaesitum; all Neof. parvum isolates caused blighting but not for all replicates of strains PD92 and PD93. DNA sequence data obtained.—For all 132 isolates six loci were sequenced and submitted to GenBank as GU251091–GU251882. A combined six-locus alignment of the DNA sequence data was submitted to TreeBase (http://purl.org/phylo/treebase/phylows/ study/TB2:S10398). Single locus analyses.—We first analyzed each of the six loci separately with parsimony (trees not shown). To speed analyses we reduced each alignment to include only one representative isolate per unique
1359
allele. (See TABLE II for descriptive statistics of the datasets and most parsimonious trees [MPTs].) Visual inspection showed that the topologies of the MPTs were similar; there was congruence with 70% bootstrap support. Minor topological differences were present within terminal clades corresponding to the morphological species B. dothidea, Neof. parvum, Neof. ribis and Neof. mediterraneum, possibly due to incomplete lineage sorting or sexual recombination (Taylor et al. 1999). In B. dothidea strain PD296 was sister of the remaining taxa based on GPD but was part of the ingroup in all other trees. Strain PD251 grouped with Neof. ribis in TUB but with Neof. parvum in HIS and HSP analyses. In Neof. mediterraneum strain PD312 was part of the ingroup in HIS and outgroup based on ITS and GPD. Six locus analyses.—Combined parsimony analyses of the six locus dataset were done with 53 unique six locus haplotypes and 3003 characters. Twenty MPTs resulted, of 2283 steps each (CI 5 0.647, RI 5 0.896) (FIG. 2). The MPTs differed by rearrangement of short branches within Neof. mediterraneum, Neof. parvum and Dip. seriata. The overall tree topology improved as compared to the single dataset analyses. Branch supports in the combined analyses were higher than for any single dataset tree at 15 branches and worse at only four branches. No single dataset tree had higher bootstrap supports than the combined analyses at more than one branch, and in those cases the combined analyses supported the branches at a minimum of 73%. Bayesian analyses yielded identical topology with generally higher branch support, except in Neos. dimidiatum, where the branching order was unresolved (tree not shown, see posterior probabilities in tree, FIG. 2). Three, four and five locus analyses.—We were interested in knowing whether the six-locus analyses provided the highest phylogenetic resolution. To investigate the effect of combinations of datasets on tree topology we compared topologies obtained by analyzing combinations of three, four and five datasets to the six locus dataset. Combined analyses of ITS, EF and TUB, commonly used in phylogenetic analyses supported 34 of the 37 nodes found in the six locus analyses with greater than 70% bootstrap support (results not shown). The combination of EF, GPD and HSP, the three datasets with the most parsimony informative sites, generated 32 supported branches. Addition of HIS, the dataset with the fourth most parsimonious sites, resulted in 32 supported branches, and addition of TUB, the fifth most informative dataset, resulted in 36 supported branches. Nodes obtained in the six locus analyses not receiving support (,70%) with fewer data were the
1360
MYCOLOGIA
TABLE II. Statistics of the single locus datasets and combined six locus dataset and respective most parsimonious trees (MPTs)
Haplotypes Characters ITS EF GPD His Hsp Tub Combined a b
35 32 33 34 36 33 53
526 405 644 543 382 503 3003
Variable characters
Parsimony informative characters
MPTs: number/steps
CI/RIb
Clades with .70% support
118 (22%)a 213 (53%) 201 (31%) 187 (34%) 187 (49%) 147 (29%) 1053/(35%)
94 (16%)a 166 (41%) 156 (24%) 151 (28%) 157 (41%) 122 (29%) 846/(28%)
2160/203 114/437 330/391 14/532 7/405 1/259 20/2283
0.759/0.892 0.730/0.856 0.673/0.874 0.545/0.751 0.657/0.838 0.726/0.868 0.647/0.896
12 18 16 18 20 16 37
Percentages refer to the proportions of variable and parsimony informative characters in each dataset. CI: consistency index; RI: retention index.
monophyly of Dip. mutila strains PD46 and PD73 (no support in combined EF, GPD, HSP and HIS dataset), monophyly of Neos. dimidiatum strains PD104 and PD105 (no support in ITS, EF, TUB), ingroup to Neof. parvum strain PD251 (no support in EF, GPD, HSP and EF, GPD, HSP, HIS), ingroup to Neof. parvum strain PD299 (no support in ITS, EF, TUB), sister group of Neof. parvum clade with strains PD17, 39, 59 (EF, GPD, HSP), Neof. parvum strains PD17, 39, 59 (no support in ITS, EF, TUB and EF, GPD, HSP), Neof. parvum strains PD17 and PD59 (no support in EF, GPD, HSP, and EF, GPD, HSP, HIS), monophyly of groups 2 and 3 (no support in EF, GPD, HSP and EF, GPD, HSP, HIS) and monophyly of groups 5 and 6 (no support in EF, GPD, HSP, HIS and EF, GPD, HSP, HIS, TUB) (results not shown). Phylogenetic groups obtained.—We obtained six 100% supported groups corresponding to clades 1–6 in Crous et al. (2006) for taxa shared between both studies (FIG. 2). Crous et al. used a 28S dataset for phylogenetic analyses of Botryosphaeriaceae and included a more diverse sample of taxa so that our results within clades are generally not directly comparable. Our branch support values were higher, possibly because we used more data. Clade 1 contained three distinct, monophyletic groups corresponding to Dip. seriata, Dip. mutila and L. theobromae respectively. Clade 2 corresponded to B. dothidea, and our analyses resolved three lineages. Lineage 1 comprised 12 isolates producing the Fusicoccum anamorph, including the B. dothidea exepitype strain PD255. Lineage 2 comprised strain PD296 with a Dichomera anamorph and lineage 3 by strain PD313 producing a Fusicoccum anamorph. Clade 3 consisted of a single isolate of M. phaseolina. Clade 4 comprised three isolates Neoscytalidium dimidiatum. Clade 5 contained three monophyletic groups corresponding to S. viticola, Do. iberica and Do. sarmentorum, each containing an ex-type strain.
Spencermartinsia viticola contained an additional relatively divergent isolate from citrus. Clade 6 was the largest and most diverse group, consisting of Neofusicoccum and Dichomera isolates. There were 10 main groups or lineages, all containing an ex-type strain, and thus corresponding to these morphological species, Neofusicoccum parvum, Neof. ribis, Neof. arbuti, Neof. andinum, Neof. nonquaesitum, Dich. eucalypti, Neof. mediterraneum, Neof. australe, Neof. lutea and Dich. versiformis. Neofusicoccum ribis was subdivided into two lineages, corresponding to the extype strain and two isolates with Dichomera states respectively. Isolates collected for this study fell into Neof. parvum, Neof. nonquaesitum, Neof. mediterraneum and Neof. australe. Neofusicoccum parvum was diverse, consisting of 23 isolates representing nine haplotypes, Neof. nonquaesitum comprised seven isolates and two haplotypes and Neofusicoccum mediterraneum consisted of 44 isolates representing six haplotypes. One isolate from blueberry grouped with Neof. australe.
TAXONOMY
Neofusicoccum nonquaesitum Inderbitzin, Trouillas, R.M. Bostock et T.J. Michailides, sp. nov. FIGS. 3–9 Conidiomata nigrae, 200–500 3 150–400 mm, conidiogenae cellulae cylindraceae, holoblastinae, proliferantes percurrentes. Conidia 23.2 3 7.6 mm, fusiformes, basi tuncata.
Holotypus: UC1946389 MycoBank MB 518135. Pycnidia single or in groups, immersed or immersed-erumpent, lenticular to subglobose, 200–500 3 150–400 mm, a short neck might be present (FIG. 3). Stroma absent or highly reduced, with loosely interwoven, pigmented hyphae associated with pycnidia at times (FIG. 3). Pycnidial wall up to 50 mm wide, three-layered. Outer layer dark, cells thickwalled, intermediate layer lighter colored, cells
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND
1361
FIG. 2. One of 20 most parsimonious trees (2283 steps each, CI 5 0.647, RI 5 0.896) inferred from the combined six locus dataset of ITS, EF, GPD, HIS, HSP and TUB (132 taxa, 3003 characters). Host and geographic origins follow strain identifiers. Bootstrap support percentages and Bayesian posterior probabilities are by the branches in that order. Branches with maximal support in both analyses are marked with asterisks. Ex-type isolates are in boldface. Morphological species and intraspecific lineages with distinctive morphological characters, such as the Dichomera states of B. dothidea and Neof. ribis, are indicated by vertical bars. Intraspecific lineages are numbered following species names. Underlined taxa occur in California. Clade affiliations are indicated by vertical bars on the right and follow Crous et al. (2006).
1362
MYCOLOGIA
FIGS. 3–9. Morphological features of Neofusicoccum nonquaesitum. 3. Vertical section of Neof. nonquaesitum strain PD484 pycnidium from inoculated bay laurel branch in the field. Microconidia marked by asterisk. 4. Squash mount of hymenium from strain PD301 grown on oak leaf agar 13 d. 5. Percurrently proliferating conidiophore of strain PD301 grown on oak leaf agar 13 d. Annelations marked by arrows. 6. Conidia of strain PD301 grown on oak leaf agar 13 d. 7. Conidia of strain PD249 grown on pistachio leaf agar 15 d. Septum marked by arrow. 8. Conidia of strain PD247 grown on pistachio leaf agar 15 d. 9. Microconidia of strain PD301 grown on oak leaf agar 13 d. Bars: 3 5 100 mm; 4–9 5 20 mm. Illumination: 3 5 phase contrast, 4– 8 5 differential interference contrast. Mounting medium: 3, 9 5 50% glycerol; 4–8 5 distilled water.
smaller, inner layer hyaline, cells thin-walled. Conidiophores short, undifferentiated, originating from the inner pycnidial wall, branching at times, up to 30 mm long, 1.5–2 mm wide, bearing single, unbranched conidiogenous cells, of similar dimensions as conidiophores (FIG. 4). Conidiogenesis holoblastic, conidiogenous cells proliferating percurrently (FIG. 5), up to five proliferations observed. Conidia hyaline, fusiform to oval, base truncate, rarely one- to threeseptate or pigmented, (17.2–)23.2 6 2.7(–29.1) 3 (5.6–)7.6 6 1.0(–10.6) mm (l/w 5 [2.3–[3.1 6 0.4 [–4.2], n 5 516) (FIGS. 6–8). Microconidia when present most abundant in upper part of pycnidium (FIG. 3), cylindrical, with rounded or truncate apices, curved at times, 4–10 3 2–4 mm, rarely up to 15 3 5 mm (FIG. 9). No chlamydospores observed.
Etymology. Negative of Latin quaesitus, meaning special, to account for the typical Neofusicoccum morphology. Cultural characteristics. Aerial mycelium on half strength PDA plate with cork oak or pistachio leaf after 12 d under continuous light on a laboratory bench white to olive-brown (1790k) or olivaceous black (2100m), reverse white to olivaceous black (2300m), pycnidia forming mainly on leaf, black, some covered by mycelium, immersed-erumpent, up to 600 mm diam and of variable shape, conidia and spermatia present. Specimens examined. UNITED STATES. CALIFORNIA: St Helena, Napa County, 38.499uN, 122.541uW, 601 m. Cankered branch of Umbellularia californica, 12 Nov 2004, F. P. Trouillas, L3IE1, PD484, CBS 126655 (culture). A sample of a branch of U. californica inoculated in the field with
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND strain PD484 was dried and deposited as UC1946389 (HOLOTYPE). (Additional specimens examined are listed in TABLE I.)
Commentary. The species description above is based on all collections of Neof. nonquaesitum in this study. Phylogenetic analyses show that Neof. nonquaesitum originating from California almond, the California native bay laurel and blueberry in Chile is most closely related to Neof. arbuti and Neof. andinum. Neofusicoccum arbuti has chlamydospores (Farr et al. 2005) that are absent in Neof. nonquaesitum, and Neof. andinum conidia are more narrow, with a length to width ratio of 4.8 (Mohali et al. 2006), as opposed to 3.1 in Neof. nonquaesitum. More than 190 species of Fusicoccum or Neofusicoccum have been described worldwide (www. indexfungorum.org). We found that of these 12 species have conidia dimensions similar to Neof. nonquaesitum (viz. mean conidia lengths 20.5–25.7 mm and length to width ratios of 2.7–3.5). Neofusicoccum luteum Pennycook & Samuels and Neof. eucalyptorum Crous, H. Sm. ter & M. J. Wingf. fall out of contention based on DNA sequence data. The remaining 10 species differ morphologically as follows: F. alocasiae N.D. Sharma (Sharma 1975), F. liriodendri (Cooke) Aa & Vanev and F. zanthoxyli (H.C. Greene) Vanev are epiphyllous (van der Aa and Vanev 2002), F. africanum van der Byl has pycnidia embedded in stromatic tissue (van der Byl 1927), F. amygdalinum (Sacc.) Ho¨hn. has conidia that measure up to 42 mm long (von Ho¨hnel 1929), F. cajani (Syd., P. Syd. & E.J. Butler) Samuels & B.B. Singh has small pycnidia with periphysoidal elements (Samuels ¨ m.) Vanev is and Singh 1986), F. dalmaticum (Thu similar morphologically to B. dothidea of which it is a synonym (Phillips et al. 2005b), F. populi A.J.L. Phillips has abundant stromatic tissue (Phillips 2000), F. tingens Goid. forms phragmospores (Goida`nich 1936), and F. yuccogena (Ellis & Everh.) Aa & Vanev pycnidia are arranged generally in groups surrounded by stromatic tissue (van der Aa and Vanev 2002). DISCUSSION
Diversity and host range of California almond isolates.— We found seven species of Botryosphaeriaceae from almond; all but B. dothidea were new records for this host. The majority of almond isolates belonged to B. dothidea, Neof. parvum, Neof. mediterraneum and Neof. nonquaesitum; a minority of isolates were Dip. seriata, Do. sarmentorum and M. phaseolina. Botryosphaeria dothidea, Neof. parvum, Neof. mediterraneum and Neof. nonquaesitum are difficult to tell apart based on anamorph morphology. We used a phylogenetic approach for species identification, defining phylogenetic species as well supported terminal or subterminal clades containing a single ex-type strain.
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Morphological identification was used for Dip. seriata and M. phaseolina because no ex-type strains were available. The occurrence of B. dothidea on almond was reported by English et al. (1966, 1975). We also found B. dothidea on California olive and blackberry, on pistachio from Greece and apple from North Carolina. In addition, preliminary results from ITS analyses showed that isolate CP4 from California pistachio from Ma et al. (2001a) belonged to B. dothidea (results not shown). Botryosphaeria dothidea is widespread; in California alone it is known from at least 42 hosts (Michailides and Morgan 2004). However this study used morphological species identification and thus should be interpreted with caution. Neofusicoccum parvum, a new record for almond, has a wide host range (Farr et al. 1989). In addition to almond we found it on walnut in California, blueberry in Chile and pistachio in Greece. Other California ´ rbez-Torres et al. 2006). hosts include grapevine (U Neofusicoccum mediterraneum found here for the first time on almond appears to be widespread and common in California. It recently was described from eucalypt in Greece and olive in Italy and previously known only from those hosts (Crous et al. 2007, Lazzizera et al. 2008). In addition we isolated Neof. mediterraneum from ash, avocado, blackberry, Citrus sp., eucalypt, juniper, kumquat, pistachio, sequoia and walnut, thus considerably expanding this species’ known host range. Neofusicoccum nonquaesitum is described as a new species in this paper. We found it on almond and California native bay laurel and also from blueberry in Chile. Diplodia seriata, Do. sarmentorum and M. phaseolina were not isolated frequently from almond. All three species are known from a variety of hosts (Farr et al. 1989). In addition to almond we found Dip. seriata on apple, peach, pistachio and walnut. In California Dip. ´ rbez-Torres et al. seriata also occurs on grapevine (U 2006) and M. phaseolina on strawberry (Koike 2008). Dothiorella sarmentorum is involved in grapevine decline in Spain (Martin and Cobos 2007). In summary our results and literature surveys indicate that none of the species found on almond are host specific but also occur on additional hosts in California as well as in other regions. Similar results have been found in South Africa where vineyards may harbor many of the same Botryosphaeriaceae species as adjacent Prunus spp. orchards (Damm et al. 2007, van Niekerk et al. 2004). Species isolated from almond band cankers.— We recorded six species from cankers. Band canker first was described by English et al. (1966), and the causal
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agent was identified as B. dothidea by English et al. (1975) based on careful comparisons to a strain of B. dothidea from North Carolina. They found that the two were indistinguishable serologically, of similar morphology and that both caused band cankers on 2 y old trees. Conidia dimensions of the almond B. dothidea isolates from English et al. (1975) were reported as 21.5 3 5.9 mm, as opposed to 24.0 3 5.7 mm for the North Carolina strain. The six species we recovered from band cankers, B. dothidea, Neof. mediterraneum, Neof. parvum, Neof. nonquaesitum, D. seriata and M. phaseolina, all were capable of inducing cankers on almond branches in field assays, accompanied by blighting of inoculated branches except for M. phaseolina. Of note, Neof. nonquaesitum and Neof. parvum, not B. dothidea, might be most virulent because the B. dothidea cankers on inoculated almond branches in the field were significantly shorter than the cankers formed by Neof. nonquaesitum and Neof. parvum (FIG. 1). These results might be mirrored by other systems. Virulence tests on grapevine, which also harbors a diverse array of Botryo´ rbez-Torres et al. 2006), sphaeriaceae species (U showed that Neof. parvum was more virulent than B. ´ rbez-Torres and Gubler 2009). But more dothidea (U research is needed to determine the relative contributions of each species to almond band canker disease. Differentiating Botryosphaeriaceae species from almond based on morphology is challenging. Diplodia seriata, with pigmented conidia, and M. phaseolina, with conidia adorned with long, unfurling polar appendages, are easily differentiated, but B. dothidea, Neof. mediterraneum, Neof. parvum and Neof. nonquaesitum all form typical Fusicoccum/Neofusicoccum pycnidia. We found that Botryosphaeria dothidea conidia were longest and most narrow (25.6 6 2.8 3 6.5 6 1.0 mm, length to width ratio [l/w] 5 4.0 6 0.7), Neof. parvum conidia were the shortest and most rounded (17.8 6 2.7 3 5 6 0.6 mm, l/w 5 2.8 6 0.6), whereas Neof. mediterraneum and Neof. nonquaesitum conidia were of intermediate dimensions and indistinguishable (Neof. mediterraneum: 22.0 6 2.0 3 7.3 6 0.7 mm, l/w 5 3.0 6 0.4; Neof. nonquaesitum: 24.3 6 2.8 3 7.6 6 1.0 mm, l/w 5 3.2 6 0.4). It is unclear what species or possibly mixture of species were observed by English et al. (1966, 1975). Macrophomina phaseolina and Dip. seriata can be ruled out, but DNA sequencing is required for conclusive identification of the remaining species. Species isolated from almond canopy cankers and other tissues.—Of the six species from band cankers B. dothidea, Neof. mediterraneum and Neof. parvum also were isolated from canopy cankers. Dothiorella
sarmentorum was only found from canopy cankers. Other almond tissues displaying disease symptoms and yielding Botryosphaeriaceae species include fruit peduncles from which B. dothidea and Neof. parvum were isolated, as well as trunk bark near the band canker that yielded Dip. seriata. The new species Neof. nonquaesitum and other potentially new species.—Our phylogenetic analyses showed that morphological and phylogenetic species concepts (Taylor et al. 2000) largely agreed because ex-type strains clustered into well supported clades. In clade 6 the well supported sister group of Neof. andinum and Neof. arbuti did not correspond to any known morphological species and thus was described as the new species Neof. nonquaesitum. Neofusicoccum nonquaesitum comprised a morphologically distinct group of 10 isolates from three hosts from California and Chile, thus letting us describe a new species based on isolates originating from different hosts and geographic origins. In some cases we found evidence of conflict between the morphological and the phylogenetic species concepts. The morphological species Neof. parvum, Neof. ribis, B. dothidea, S. viticola and L. theobromae contained distinct lineages, possibly cryptic species, consisting of a single isolate or a small group of well supported isolates that differed from ex-type strains. However, in all cases we think that more isolates are needed before any taxonomic conclusions can be drawn. Neofusicoccum parvum received support for monophyly only in the Bayesian analyses and comprised at least four different lineages, each receiving greater than 70% support in both parsimony and Bayesian analyses. With five loci Pavlic et al. (2009a) identified three cryptic lineages related to Neof. ribis and Neof. parvum from a single tree host in South Africa and designated them new species (Pavlic et al. 2009b). We did not include their new species, but combined analyses of data generated in this study with the data of Pavlic et al. showed that our lineages were different (results not shown). We were unable to find any distinctive morphological characters for the four lineages (results not shown) similar to Pavlic et al. (2009b). Strains with Dichomera anamorphs attributed to B. dothidea and Neof. ribis earlier (Barber et al. 2005) were phylogenetically divergent. They differed by nearly 20 substitutions at the six loci from their respective closest relatives. This genetic differentiation reflects morphological divergence because none of the other 13 B. dothidea and Neof. ribis isolates examined formed the Dichomera state (FIGS. 10–14). Elevating the Dichomera lineage of B. dothidea represented by strain PD296 to species would result in paraphyly of B. dothidea and thus would require introduction of additional species,
INDERBITZIN ET AL.: BOTRYOSPHAERIACEAE FROM ALMOND
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FIGS. 10–16. Morphological features of other new lineages recovered in this study. 10, 11. Fusicoccum and Dichomera conidia of B. dothidea strain PD296 grown on peach leaf agar 13 d. 12. Fusicoccum conidia of B. dothidea strain PD313 grown on peach leaf agar 12 d. No Dichomera conidia were observed. 13, 14. Dichomera conidia of Neof. ribis strain PD288 grown on peach leaf agar 18 d. No Neofusicoccum conidia were observed. 15, 16. Conidia of S. viticola strain PD74 grown on oak leaf agar 13 d. Optical sections of one conidium showing the smooth inner wall and the furrowed outer wall, unlike the type of S. viticola. Bars: 10–14 5 20 mm; 15, 16 5 10 mm. Illumination: 10–14 5 differential interference contrast. Mounting medium: 10– 16 5 distilled water.
for instance for the lineage represented by strain PD313 (FIG. 12). Barber et al. (2005) also described a Dichomera state of Neof. parvum and Neof. australe of which we were unable to obtain any representatives. The parallel formation of Dichomera states accompanied with genetic differentiation in at least two instances is interesting and deserves further studies with a larger dataset than we had access to. Another distinct lineage was present within S. viticola by strain PD74 that differed morphologically from the type of S. viticola by its conidia whose walls are furrowed on the outside and smooth on the inside (FIGS. 15, 16), unlike the type (Luque et al. 2005). The two strains of L. theobromae included in these studies differed considerably genetically. Alves et al. (2008) identified three lineages within L. theobromae and described two new species. But until there is an ex-type culture of L. theobromae available for DNA sequencing the taxonomic situation in this group will remain ambiguous.
The remaining species contained minor molecular variation; these include Neof. mediterraneum found from a variety of hosts in California and also known to occur in Europe (Crous et al. 2007). Single phylogenetic trees differed in their topology for Neof. mediterraneum, indicative of gene re-assortment during meiosis, yet no sexual state is known for this species. Phylogenetic resolution of different dataset combinations.—We found that combined analyses of all six loci resulted in the highest phylogenetic resolution. Combined analyses of ITS, EF and TUB, widely used for phylogenetics in Botryosphaeriaceae, resulted in 34 nodes with greater than 70% bootstrap support, as compared to the 37 for the six locus analyses. However six loci were critical within the diverse Neof. parvum; five of the nine nodes not supported by any of the three, four or five locus combined analyses were located in Neof. parvum. Whether a saturation point of phylogenetic resolution for our taxon sample
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has been reached is unclear. There is no evidence that the number of well supported nodes had leveled off because the five locus combination of EF, GPD, HIS, HSP and TUB supported 36 nodes, one fewer than in the six locus combined analyses. More data could be added until the number of supported nodes fails to increase, which depends on the particular phylogenetic circumstances at hand (Gatesy et al. 2007). CONCLUSION
Our survey of Botryosphaeriaceae species centered on almond in California resulted in six new records for this host, suggesting that in addition to B. dothidea other species might be involved in almond band canker formation. Neofusicoccum nonquaesitum and Neof. parvum appeared to be more virulent than B. dothidea when inoculated on almond branches in the field, but more research is necessary to assess the contributions of these species to band canker disease. All species recovered from almond also occurred on other California hosts, which has implications on band canker epidemiology and disease management. Despite intensive research in Botryosphaeriaceae systematics in recent years, we found one new species, Neof. nonquaesitum, from several hosts including almond and bay laurel in California and blueberry in Chile, suggesting that this species might be widely distributed and could have a wide host range. More species likely are awaiting characterization in Botryosphaeriaceae because we found evidence for the presence of new lineages correlating with morphological characters. These are the Dichomera anamorphic state in B. dothidea and Neof. ribis and conidial wall characteristic in S. viticola. Despite using six loci, more than most previous studies in this group of fungi, we found evidence that adding more data might improve phylogenetic resolution. ACKNOWLEDGMENTS
We thank Mike Wingfield and Bernard Slippers, FABI, University of Pretoria, Nuccia Eyres, Department of Agriculture, government of Western Australia, Turner Sutton, North Carolina State University, and Bernardo Latorre, Pontificia Universidad Cato´lica de Chile, for providing cultures critical to this work, Dave Morgan, UC Davis, for suggestions that improved the manuscript, and the California Almond Board for financial support.
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